Recombination‐Based Assay (RBA) for Screening Bacteriophage Lambda Libraries

David M. Kurnit1

1 University of Michigan Medical Center, Ann Arbor, Michigan
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.12
DOI:  10.1002/0471142727.mb0612s27
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The recombination‐based assay represents a convenient way to screen a complex library constructed in bacteriophage l for homology to a given sequence cloned into a specially designed plasmid. The technique serves to screen a bacteriophage library rapidly and efficiently with a sequence cloned into a plasmid; counterselection then yields the gene product of interest with its plasmid carrier deleted. Because 106 to 107 plaque‐forming units (pfu) may be screened using several petri dishes, and the homology for crossing‐over need only be greater than 25 bp, the RBA represents an efficient way to screen complex l libraries rapidly for homology to a given sequence. In this procedure, a l library is screened using a specially designed plasmid carrying the desired target sequence. Recombinants arising from cross‐over events between the plasmid and a bacteriophage carrying a corresponding region of homology are selected by their ability to grow on strain DM21. Growth of l on DM21 requires the presence of an allele encoded on the plasmid to suppress an amber mutation in the host strain that prevents l propagation. Recovery of the original phage carrying the target sequence requires a reversal of the homologous recombination event. This reversal occurs spontaneously, and is detected by PCR amplification using primers that flank the cloning site in the l vector.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 4.
  • DNA fragment encoding sequence of interest
  • Plasmid pAD1 (Fig. ; available from Dr. D. Kurnit)
  • recA+E. coli strain (Table 97.80.4711 or commercial suppliers)
  • recipeL broth (see recipe) with 50 µg/ml kanamycin (Table 97.80.4711)
  • Bacteriophage λ library (unit 5.8)
  • recipeLambda top agar (see recipe)
  • recipeLambda plates (see recipe), some with 50 µg/ml kanamycin and some with 100 µg/ml streptomycin (Table 97.80.4711)
  • recipeSuspension medium (SM; see recipe)
  • Chloroform
  • E. coli DM21, DM75, DM392, and DM1061 (Fig. and Table 6.12.1), saturated overnight cultures freshly grown in LB medium (unit 1.1) with 100 µg/ml streptomycin
    Table 6.2.1   MaterialsBacterial Strains Used

    Strain Genotype a Reference Comment
    DM21 lacZ YA536(am), dnaB266(am), Smr, hsdR+, hsdM+, ?tonAimm21 b 515 b519 nin5 att+ P1ban) Kurnit and Seed, 1990 sup0 lacZam; dnaBam
    DM75 lacZ YA536(am), Smr, hsdR+,, hsdM+, ?tonA Guarente et al., sup0 lacZam strain used by the author and collaborators is Smr, although the published genotype does not state this
    DM392 hsdR514 (hsdR, hsdM+), supE44, supF58, ?lacY1, galK2, galT22, metB1, trpR 55, Smr, ?tonA L. Enquist (unpub. observ.) sup+; made Smr by the author and collaborators
    DM1061 araD139, Δ(ara, leu)7697, ΔlacX74, galU, galK, Smr, hsdR, hsdM+, mcrA,mcrB, ?tonA Casadaban and Cohen, sup0

     aA question mark denotes characteristics that are likely, but not definite.
  • 100 mM IPTG (isopropyl thiogalactoside; Table 97.80.4711)
  • recipe2% Xgal in DMF (see recipe)
  • Additional reagents and equipment for subcloning DNA into plasmids (unit 3.16), culturing (unit 1.1) and transformation (unit 1.8) of bacteria, plating and titering λ phage (unit 1.11), β‐galactosidase assay (unit 1.4), and PCR amplification (unit 15.1)
NOTE: All incubations are at 37°C unless otherwise specified.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bolivar, F., Rodriguez, R., Green, P.J., Betlach, M., Heyneker, H.L., Boyer, H.W., Crosa, J., and Falkow, S. 1977. Construction and characterization of new cloning vehicles. Gene 2:95‐113.
   Buckler, A.J., Chang, D.D., Graw, S.L., Brook, J.D., Haber, D.A., Sharp, P.A., and Housman, D.E. 1991. Exon amplificaton: A strategy to isolate mammalian genes based on RNA splicing. Proc. Natl. Acad. Sci. U.S.A. 88:4005‐4009.
   Casadaban, M.J. and Cohen, S.N. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179‐207.
   Duyk, G.M., Kim, S., Myers, R.M., and Cox, D.R. 1990. Exon trapping: A genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA. Proc. Natl. Acad. Sci. U.S.A. 87:8995‐8999.
   Guarente, L., Lauer, G., Roberts, T.M., and Ptashne, M. 1980. Improved methods for maximizing expression of a cloned gene: A bacterium that synthesizes rabbit β‐globin. Cell 20:543‐553.
   Hanzlik, A.J., Hauser, M.A., Osemlak‐Hanzlik, M.M., and Kurnit, D.M. 1993. The recombination‐based assay demonstrates that the fragile X sequence is transcribed widely during development. Nature Genet. 3:44‐48.
   Hochgeschwender, U. 1994. Identifying transcribed sequences in arrayed bacteriophage or cosmid libraries. In Current Protocols in Human Genetics (Dracopoli, N., Haines, J.L., Korf, B., Moir, D.T., Morton, C.M., Seidman, C.E., Seidman, J.G., and Smith, D.R., eds.) pp. 6.2.1‐6.2.15. John Wiley & Sons, New York.
   Hochgeschwender, U., Sutcliffe, J.G., and Brennan, M.D. 1989. Construction and screening of a genomic library specific for mouse chromosome 16. Proc. Natl. Acad. Sci. U.S.A. 86:8482‐8486.
   Huynh, T., Young, R.A., and Davis, R.W. 1985. Constructing and screening cDNA libraries in λgt10 and λgt11. In DNA cloning, Vol. II (D. Glover, ed.) IRL Press, Eynsham, U.K.
   Ikeda, H., Aoki, K., and Naito, A. 1982. Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: Structure of recombinant DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 79:3724‐3728.
   Jankowski, S., Stewart, G.D., Buraczynska, M., Galt, J., Van Keuren, M., and Kurnit, D.M. 1990. Molecular approaches to trisomy 21. Prog. Clin. Biol. Res. 360:79‐88.
   Kao, F.‐T. and Yu, J.‐W. 1991. Chromsome microdissection and cloning in human genome and genetic disease analysis. Proc. Natl. Acad. Sci. U.S.A. 88:1844‐1848.
   King, S.R. and Richardson, J.P. 1986. Role of homology and pathway specificity for recombination between plasmids and bacteriophage λ. Mol. Gen. Genet. 204:141‐147.
   Kurachi, S., Baldori, N., and Kurnit, D.M. 1989. Sumo 15A: A lambda plasmid that permits easy selection for and against cloned inserts. Gene 85:35‐43.
   Kurnit, D.M. and Seed, B. 1990. Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo. Proc. Natl. Acad. Sci. U.S.A. 87:3166‐3169.
   Lawn, R.M., Fritsch, E.H., Parker, R.C., Blake, G., and Maniatis, T. 1978. The isolation and characterization of linked δ‐ and β‐globin genes from a cloned library of human DNA. Cell 15:1157‐1174.
   Liu, P., Legerski, R., and Siciliano, M.J. 1989. Isolation of human transcribed sequences from human‐rodent somatic cell hybrids. Science 246:813‐815.
   Lovett, M. 1994. Direct selection of cDNAs using genomic contigs. In Current Protocols in Human Genetics (N. Dracopoli, J.L. Haines, B. Korf, D.T. Moir, C.M. Morton, C.E. Seidman, J.G. Seidman, and D.R. Smith, eds.) pp. 6.3.1‐6.3.15. John Wiley & Sons, New York.
   Lovett, M., Kere, J., and Hinton, L.M. 1991. Direct selection: A method for the isolation of cDNAs encoded by large genomic regions. Proc. Natl. Acad. Sci. U.S.A. 88:9628‐9632.
   Lutz, C.T., Hollifield, W.C., Seed, B., Davie, J.M., and Huang, H.V. 1987. Syrinx 2A: An improved λ phage vector designed for screening DNA libraries by recombination in vivo. Proc. Natl. Acad. Sci. U.S.A. 84:4379‐4383.
   Marvo, S.L., King, S.R., and Jaskunas, S.R. 1983. Role of short regions of homology in intermolecular illegitimate recombination events. Proc. Natl. Acad. Sci. U.S.A. 80:2452‐2456.
   Neve, R.L. and Kurnit, D.M. 1983. Comparison of sequence repetitiveness of human cDNA and genomic DNA using the miniplasmid vector piVX. Gene 23:355‐367.
   Neve, R.L., Bruns, G.A.P., Dryja, T.P., and Kurnit, D.M. 1983. Retrieval of human DNA from rodent‐human genomic libraries by a recombination process. Gene 23:343‐354.
   Nisson, P.E. and Watkins, P.C. 1994. Isolation of exons from cloned DNA by exon trapping. In Current Protocols in Human Genetics (Dracopoli, N., Haines, J.L., Korf, B., Moir, D.T., Morton, C.M., Seidman, C.E., Seidman, J.G., and Smith, D.R., eds.) pp. 6.1.1‐6.1.14. John Wiley & Sons, New York.
   Parimoo, S., Patanjali, S.R., Shukla, H., Chaplin, D.D., and Weissman, S.M. 1991. cDNA selection: Efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments. Proc. Natl. Acad. Sci. U.S.A. 88:9623‐9627.
   Poustka, A., Rackwitz, H.‐R., Frischauf, A., Hohn, B., and Lehrach, H. 1984. Selective isolation of cosmid clones by homologous recombination in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 81:4129‐4133.
   Rubin, C.M., Houck, C.M., Deininger, P.L., and Schmid, C.W. 1980. Partial nucleotide sequence of the 300 nucleotide interspersed repeated human DNA sequences. Nature 284:372‐374.
   Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G., Erlich, H.A., and Arnheim, N. 1985. Enzymatic amplification of β‐globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350‐1354.
   Seed, B. 1983. Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. Nucl. Acids Res. 11:2427‐2445.
   Shen, P. and Huang, H.V. 1986. Homologous recombination in Escherichia coli: Dependence on substrate length and homology. Genetics 112:441‐457.
   Shen, P. and Huang, H.V. 1989. Effect of base pair mismatches on recombination via the recBCD pathway. Mol. Gen. Genet. 218:358‐360.
   Short, J.M., Fernandez, J.M., Sorge, J.A., and Huse, W.D. 1988. λ ZAP: A bacteriophage λ expression vector with in vivo excision properties. Nucl. Acids Res. 16:7583‐7599.
   Stewart, G.D., Hauser, M.A., Kang, H., McCann, D.P., Osemlak, M.M., Kurnit, D.M., and Hanzlik, A.J. 1991. Plasmids for recombination‐based screening. Gene 106:97‐101.
   Watt, V.M., Ingles, C.J., Urdea, M.S., and Rutter, W.J. 1985. Homology requirements for recombination in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 82:4768‐4772.
   Yanisch‐Perron, C., Vieira, J., and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103‐119.
   Young, R.A. and Davis, R.W. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. U.S.A. 80:1194‐1198.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library